Biomedical subjects

J A Jarvis

Publications and source records attributed to J A Jarvis.

At least 19 recordsLinked to original sources

PubMed1995

Solution structure of the acetylated and noncleavable mitochondrial targeting signal of rat chaperonin 10.

Chaperonin 10 (Cpn10) is one of only a few mitochondrial matrix proteins synthesized without a cleavable targeting signal. Using a truncated form of Cpn10 and synthetic peptides in mitochondrial import assays, we show that the N-terminal region is both necessary and sufficient for organellar targeting in vitro. To elucidate the structural features of this topogenic signal, peptides representing residues 1-25 of rat Cpn10 were synthesized with and without the naturally occurring N-terminal acetylation. 1H NMR spectroscopy in 20% CF3CH2OH,H2O showed that both peptides assume a stable helix-turn-helix motif and are highly amphiphilic in nature. Chemical shift and coupling constant data revealed that the N-terminal helix is stabilized by N-acetylation, whereas NOE and exchange studies were used to derive a three dimensional structure for the acetylated peptide. These findings are discussed with respect to a recent model predicting that targeting sequences forming a continuous alpha-helix of more than 11 residues cannot adopt a conformation necessary for proteolysis by the matrix located signal peptidases (Hammen, P. K., Gorenstein, D. G., and Weiner, H. (1994) Biochemistry 33, 8610-8617).

Acetylation↗

PubMed1995

13C NMR relaxation studies of molecular motion in peptide fragments from human transthyretin.

Natural-abundance 13C T1 and NOE measurements have been made for backbone and side-chain sites in peptide fragments of transthyretin (TTR 10-20, TTR 105-115, and TTR 105-115Met111) at 13C Larmor frequencies of 125 and 75 MHz. These peptides have previously been implicated in the formation of amyloid fibrils. The data were systematically assessed for their consistency with theoretical relaxation parameters derived from models of molecular motion. It was shown that of four models, ranging from simple isotropic motion to one defining internal wobbling of the 13C-1H vector, the "model-free approach" (Lipari and Szabo, J. Am. Chem. Soc. 104, 4546, 1982) was best able to predict the experimental data. These peptides exhibited overall correlation times close to 1 ns. Internal motions with effective correlation times of approximately 0.08 ns were observed for backbone carbon sites, and side-chain carbons exhibited more rapid and less ordered motions. No indication of retarded motion due to the presence of small peptide aggregates was detected, in agreement with reports of the rapid incorporation of these species into amyloid fibrils.

Amino Acid Sequence↗

PubMed1994

Structural analysis of peptide fragment 71-93 of transthyretin by NMR spectroscopy and electron microscopy: insight into amyloid fibril formation.

A peptide corresponding to the amino acid region 71-93 of the plasma protein transthyretin (TTR) has been synthesized to investigate its role in the native folding of the molecule and the possible relationship between mutations in this region and amyloid formation of TTR. In the native structure this fragment includes a beta-strand followed by a short helix and turns back on itself to form part of an antiparallel beta-sheet. Electron microscopy has shown that the peptide is not intrinsically amyloidogenic. NMR spectroscopy has been used to investigate the conformational dependency of the peptide on the solution conditions. Minor populations of peptide showing partial turns were apparent in deuterated dimethyl sulfoxide (DMSO-d6). Some indication of nascent helix between residues 5 and 12 was observed in water, and upon the addition of 20% trifluoroethanol (TFE) the span of helix was confirmed. The intrinsic tendency to form a helical structure between residues 5 and 12 in solution suggests that the helical region, also present in the native crystallographically determined TTR structure at corresponding residues 75-82, is an important folding initiation site. In contrast, the beta-sheet motif observed in the native structure was not detected in solution. It is proposed that mutations in TTR occurring in the helical region result in subtle changes in the TTR structure leading to amyloid fibril formation.

Amino Acid Sequence↗

PubMed1994

1H NMR analysis of fibril-forming peptide fragments of transthyretin.

Peptide fragments of the protein transthyretin, previously shown to form cross beta-sheet amyloid-like fibrils in vitro, were investigated using 1H 1D and 2D NMR techniques. TTR 10-20, TTR 105-115 as well as a substituted analogue, (TTR 105-115Met111) all formed amyloid-like fibrils readily in 20-30% acetonitrile/water at room temperature. It was found that the presence of fibrils in the peptide solutions did not affect the observable NMR spectra, which may have been due to the line-broadening that would be associated with these macromolecular species. 1H NMR spectra were thus representative of the monomeric form of the peptide in solution. Information from D2O exchange, 3JNH-alpha H coupling measurements, temperature coefficients and NOESY experiments suggested that these peptides have some propensity for turn or helix but were predominantly unstructured. There was no indication of the monomeric species existing predominantly in an extended form, suggesting that the formation of beta-sheet based fibrils does not require preformed extended structures. TTR 105-115Met111 displayed slight structural differences from TTR 105-115 which may be related to the fibril-forming propensity of the corresponding mutant TTR.

Amino Acid Sequence↗

PubMed1993

X-ray diffraction studies of fibrils formed from peptide fragments of transthyretin.

Two synthetic peptide fragments of the plasma protein transthyretin (TTR), previously shown to form fibrillar structures in vitro, have been examined using electron microscopy and X-ray diffraction. The fibrils displayed all characteristics of cross beta-sheet conformation with antiparallel strand spacing of 4.7 A and intersheet spacings of 8-10 A as well as reflections indicating further lateral repeating units. A third peptide containing a substitution equivalent to a mutation in TTR known to increase the propensity of TTR to form amyloid was also examined. It also formed fibrils and showed similar cross beta-sheet structure, but with closer intersheet packing than its native equivalent.

Amino Acid Sequence↗

PubMed1992

Homology model of thyroxine binding globulin and elucidation of the thyroid hormone binding site.

The tertiary structure of thyroxine binding globulin (TBG) has been modelled on the basis of its close homology to alpha 1-antitrypsin, the archetype of the serine protease inhibitor (serpin) superfamily. Energy minimization was applied to the model to refine the structure further. The putative thyroid hormone binding region suggested in previous labelling studies was found to exist within a beta-barrel structure of complementary dimensions to the thyroid hormones. The model also revealed that the binding cleft provides the hydrophobic environment and specific ionic interaction sites deemed important for thyroid hormone binding. The model is in good agreement with evidence derived from previously reported T3 and T4 binding, stability and isoelectric focussing studies of TBG and TBG variants. Finally, T4 analogue and drug binding studies have enabled us to postulate the orientation and manner of hormone binding to TBG. This may prove to be of assistance in the development of potent and specific, non-thyroidal ligands and also aid in the understanding of physiological thyroid hormone binding interactions.

Amino Acid Sequence↗

PubMed1984

In vivo inhibition of dopamine-beta-hydroxylase in rat adrenals during exposure to carbon disulphide.

Male rats were exposed for a maximum of 4 h to carbon disulphide at atmospheric levels of 1.0-4.0 mg/l and the turnover rates of adrenal dopamine was determined by injecting alpha-methyl-p-tyrosine and measuring the rate at which dopamine disappears. Although the level of exposure was significantly higher than the 30.0 micrograms/l permissible limit, or the average occupational exposure, similar or even higher peak exposure values were reported from the viscose rayon industry. After inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine, adrenal dopamine contents declined at a slower rate in rats exposed to carbon disulphide than in controls. The reduced rate of dopamine metabolism during exposure to carbon disulphide indicates inhibition of dopamine-beta-hydroxylase in vivo. The size of this effect, which could be detected as soon as 30 min after starting the exposure to carbon disulphide, was dose dependent. The rate of dopamine turnover was still reduced 2 h after the end of a single exposure. However at that time, because of the larger dopamine pool present in the adrenals, the amount of dopamine converted per unit of time was again at pre-exposure levels.

Adrenal Glands↗

PubMed1981

The inhibitory effect of serum from diethyldithiocarbamate treated rats on bovine adrenal dopamine-beta-hydroxylase activity.

The activity of serum dopamine-beta-hydroxylase does not depend solely on the presence of exogenous inhibitors: its activity also depends on the rate of release into plasma and is influenced by sympathetic activity or pathological conditions. This fluctuation may disguise the effect of an exogenous inhibitor on the enzyme. It has been shown in the present work that boiling of serum obtained from sodium diethyldithiocarbamate treated rats did not eliminate the dopamine-beta-hydroxylase inhibitory effect on a bovine adrenal dopamine-beta-hydroxylase preparation. Thus it is possible to measure the dopamine-beta-hydroxylase inhibitory effect of an exogenous inhibitor of this type without interference from the enzyme present in serum. Serum taken from rats treated with sodium diethyldithiocarbamate depressed in a time and dose dependent way the activity of bovine adrenal dopamine-beta-hydroxylase preparation.

Adrenal Glands↗

PubMed1976

Effects of diethyldithiocarbamate on the catabolism of tyrosine in the rat brain.

500 mg/kg sodium diethyldithiocarbamate (DDC) and trace quantities of uniformly labelled 14C-tyrosine were administered simultaneously to male albino rats of Porton-Wistar strain of approximately 210 g body weight. Thirty minutes later total radioactivity, the concentration and the specific activity of free tyrosine were increased both in plasma and in the brain by 40% compared with rats untreated with DDC. The incorporation of 14C from 14C-tyrosine into the fraction corresponding to the elution of glutamine-glutamate from the amberlite resin column was 80% less in the brain 30 min after DDC. The exhalation of 14CO2 was depressed by 80% in the first hour after DDC. When 14C-tyrosine was given 3.5 h after DDC the only differences between experimental and control rats were the increased incorporation of 14C into the glutamine-glutamate and aspartate fractions and the increased exhalation of 14CO2 which became significiant in the third and fourth half hour periods after the injection of 14C-tyrosine. From the experiments it is concluded that DDC, an inhibitor of dopamine-beta-hydroxylase, also interferes with the major catabolic pathway of tyrosine.

Animals↗

PubMed1974